Insulin-like growth factor-1 (IGF-1; a 70 amino-acid polypeptide) and insulin-like growth factor-2 (IGF-2; a 67 amino-acid polypeptide) are 7.5-kD soluble factors present in serum that can potently stimulate the growth of many mammalian cells (reviewed by Pollack et al., 2004). On secretion into the bloodstream the IGFs form complexes with the IGFBPs which protect them from proteolytic degradation in the serum en route to their target tissues and prevents their association with the IGF receptors. IGFs are also known to be secreted in an autocrine or paracrine manner in target tissues themselves. This is known to occur during normal fetal development where the IGFs play a key role in the growth of tissues, bone and organs. It is also seen in many cancer tissues where there is thought to be paracrine signaling between tumour cells and stromal cells or autocrine IGF production by the tumour cells themselves (reviewed by LeRoith D, 2003).
IGF-1 and IGF-2 are able to bind to the IGF-1 receptor (IGF-1R) expressed on many normal tissues, which functionally is a 460 kD heterotetramer consisting of a dimerised alpha- and beta-subunit, with similar affinities (Rubin et al., 1995). IGF-2 can also bind to the IGF-2 receptor, which is thought to prevent IGF-2 from binding and signaling through the IGF-1R. In this respect the IGF-2R has been demonstrated to be a tumour suppressor protein. The IGF-1R is structurally similar to the insulin receptor which exists in two forms, IR-A and IR-B, which differ by an alternatively spliced 12 amino acid exon deletion in the extracellular domain of IR-A. IR-B is the predominant IR isoform expressed in most normal adult tissues where it acts to mediate the effects of insulin on metabolism. IR-A on the other hand is known to be highly expressed in developing fetal tissues but not in adult normal tissues. Recent studies have also shown that IR-A, but not IR-B, is highly expressed in some cancers. The exon deletion in IR-A has no impact on insulin binding but does cause a small conformational change that allows IGF-2 to bind with much higher affinity than for IR-B (Frasca et al., 1999; Pandini et al., 2002). Thus, because of it's expression in cancer tissues and increased propensity for IGF-2 binding, IR-A may be as important as IGF1-R in mediating the mitogenic effects of IGF-2 in cancer.
Binding of the IGFs to IGF-1R triggers a complex intracellular signaling cascade which results in activation of proteins that stimulate proliferation and survival (reviewed by Pollack et al., 2004).
Unlike the EGFR and Her2neu receptors there is no known amplification of the IGF1-R or IR-A receptors in cancers indicating that receptor activation is controlled by the presence of active ligand. There is a very large body of scientific, epidemiological and clinical literature implicating a role for the IGFs in the development, progression and metastasis of many different cancer types (reviewed by Jerome et al., 2003; and Pollack et al., 2004).
For example, in colorectal cancer the expression of IGF-2 mRNA and protein is elevated in clinical colorectal tumour specimens compared with adjacent normal tissue (Freier et al., 1999; Li et al., 2004). There is also a positive correlation of elevated IGF serum levels with proliferating cell index in patients with colorectal neoplasia (Zhao et al., 2005). In addition, elevated circulating levels of IGF-2 correlate with an increased risk of developing colorectal cancers and adenomas (Renehan et al., 2000a) and b); Hassan et al., 2000). Loss of parental imprinting (LOI) of the IGF-2 gene, an epigenetic alteration that results in elevated IGF-2 expression, is a heritable molecular trait that has recently been identified in patients with colorectal and other tumour types. Loss of IGF-2 imprinting has been shown to be associated with a five-fold risk of colorectal neoplasia (Cui et al., 2003; Cruz-Correa et al., 2004) and adenomas (Woodson et al., 2004). Antibodies targeting the alpha-subunit of the IGF-1R which block IGF binding and internalize the receptor have been shown to delay the growth of the xenografted colon cancer-derived cell lines such as COLO 205 (Burtrum et al., 2003).
Elevated levels of IGFs are associated with a poor prognosis in human pulmonary adenocarcinomas (Takanami et al., 1996) and IGFs are expressed and secreted by many SCLC- and NSCLC-derived cell lines (Quinn et al., 1996). Transgenic over-expression of IGF-2 induces spontaneous lung tumours in a murine model (Moorhead et al., 2003). In terms of hepatocellular carcinoma (HCC), human clinical specimens and animal models of HCC express higher levels of IGF mRNA and protein than corresponding normal tissues and this has been correlated with increased tumour growth (Wang et al., 2003; Ng et al., 1998). IGF-2 has also been shown to be a serological marker of HCC with elevated levels in the serum of HCC patients compared with controls (Tsai et al., 2005).
Many childhood solid tumours such as Ewing's sarcoma and rhabdomyosarcoma appear to be particularly dependent on the IGF signaling pathway for their growth (Scotlandi et al., 1996). LOI of the IGF-2 gene has been implicated as a primary genetic event in the development for embryonal rhabdomyosarcoma (Fukuzawa et al., 1999). Autocrine IGF signaling is also thought to strongly influence the growth of Ewing's sarcoma in cases where the type-1 EWS-FLI1 chimeric transcription factor is expressed through a chromosomal translocation resulting in elevated expression of target genes including the IGF ligands and IGF-1R, and reduced expression of IGFBP-3. Antibodies and small molecule compounds targeting the IGF-1R have been shown to reduce the growth of xenografted pediatric solid tumour derived cell lines (Kolb et al., 2008; Manara et al., 2007).
Using IGF ligand-specific antibodies it has been demonstrated that the growth of human prostate cancer cells in adult human bone implanted into SCID mice can be inhibited (Goya et al., 2004). In addition, it was demonstrated that the same IGF ligand antibodies could block the paracrine supply of IGF and suppress the liver metastasis of human colorectal cancer cells in a murine xenograft system (Miyamoto et al., 2005).
There is also considerable evidence suggesting that the IGF signaling system reduces the sensitivity of cancers to chemotherapeutic agents and radiation. One of the earliest findings in this respect was the demonstration that IGF-1R knock-out mouse embryos are refractory to transformation by viruses, oncogenes and over-expressed growth factor receptors (Sell et al., 1993; Sell et al., 1994) and that over-expression of IGF-1R protects cells from UV irradiation and gamma radiation-induced apoptosis (Kulik et al., 1997). Furthermore, using liver tumour cell lines that secrete large amounts of IGF-2, it was found that neutralization of IGF-2 significantly increased response to chemotherapeutic agents such as cisplatin and etoposide in vitro, especially at lower, cytostatic doses, suggesting that IGF-2 can reduce the susceptibility to chemotherapeutic agents (Lund et al., 2004). Consistent with these findings it has been demonstrated that antibodies targeting the IGF-1R increase the susceptibility of tumour xenografts to growth inhibition by chemotherapeutic drugs and radiation (Goetsch et al., 2005).
A number of antibodies that show cross-reactive binding to human IGF-1 and human IGF-2 have been reported. Antibody sm1.2 was raised against human IGF-1 and shows 40% cross-reactivity to human IGF-2 and was shown to inhibit the proliferation of a mouse fibroblast cell line BALB/c3T3 which was stimulated with 20 ng/ml human IGF-1 (Russell et al., 1984). In a study designed to functionally epitope map IGF-1 by raising monoclonal antibodies to whole IGF-1 protein and portions of the protein a number of antibodies where identified that cross reacted with IGF-2 (Manes et al., 1997). The percent cross-reactivity with IGF-2 ranged from 0 to 800% and several antibodies were identified which were equally IGF-1 and IGF-2 reactive. KM1486 is a rat monoclonal antibody that cross-reacts with human IGF-1 and IGF-2 and it was demonstrated that KM1486 can inhibit growth of human prostate cancer cells in human adult bone implanted into nonobese diabetic/severe combined immunodeficient mice (Goya et al., 2004). In addition, it was demonstrated that KM1486 suppresses the liver metastasis of human colorectal cancers (Miyamoto et al., 2005). KM1486 has also been described in WO 03/093317, JP 2003-310275, WO 2005/018671, WO 2005/028515, and WO 2005/027970.
For the treatment of human disease an antibody with a fully human sequence is highly desirable in order to minimize the risk of generating a human anti-antibody reaction and neutralizing antibodies that will rapidly eliminate the administered antibody from the body and thereby reduce the therapeutic effect. As such, and given the roles of IGF-1 and IGF-2 dependent signaling in the development and progression of cancers, it has become desirable to obtain fully human antibodies. WO 2007/070432 describes fully human antibodies that co-neutralise the mitogenic effects of both ligands.
It was an object of the invention to provide alternative human anti-IGF antibodies with high affinities.
It was a further object of the invention to provide human anti-IGF antibodies with high affinity to IGF-1.
It was a further object of the invention to provide human anti-IGF antibodies with high affinity to IGF-1 and to IGF-2.
It was a further object of the invention to provide human anti-IGF antibodies with adequate relative affinities to IGF-1 and to IGF-2.
It was a further object of the invention to provide human anti-IGF antibodies with a higher affinity to IGF-1 than to IGF-2.
It was a further object of the invention to provide human anti-IGF antibodies with high IGF-1 neutralisation potency.
It was a further object of the invention to provide human anti-IGF antibodies with high IGF-1 and IGF-2 neutralisation potency.
It was a further object of the invention to provide human anti-IGF antibodies with high solubility and stability.
It was a further object of the invention to obtain antibodies that do not affect binding of insulin to its receptor.
The clinical development of therapeutic agents is supported by pharmacodynamic biomarkers of drug activity. Clinical studies with antibodies targeting the IGF-1R have demonstrated that an increase in total serum IGF-1 levels may be a useful pharmacodynamic marker for these agents (Pollack et al., 2007). The reason for the increase in total serum IGF-1 levels is likely due to a feedback mechanism involving pituitary growth hormone (GH) secretion which releases both IGF-1 and IGFBPs from the liver. Indeed, in humans it has been demonstrated that free or bioactive IGF-1, which represents only around 1% of total IGF-1 levels, determines the feedback response (Chen et al., 2005).
It was therefore a further object of the invention to provide, for the treatment of diseases in whose development and/or progression the IGFs are causally involved, a therapy that is accompanied by a biomarker that allows the pharmacological monitoring of the effectiveness of the therapy.
In the experiments of the present invention, it could be demonstrated that total serum IGF-1 levels are elevated upon application of the anti-IGF antibodies of the invention. Thus, total IGF-1 levels are a useful pharmacodynamic marker for the effectiveness of the therapy with an anti-IGF antibody. It is therefore highly advantageous that the antibodies of the invention are cross-reactive with IGFs from a suitable animal species, e.g. mouse or rat, such that a pharmacodynamic effect can already be tested pre-clinically.
“Total IGF-1 levels” refers to the combined amount of IGF-1 in plasma or serum comprising the amount of IGF-1 bound to serum binding proteins plus the free (unbound) IGF-1.
Therefore, in a further aspect, the present invention relates to a method for determining the effectiveness of a treatment of a cancer patient with an antibody molecule that binds to IGF-1 and IGF-2. In such method, in a first step, the level of total IGF-1 is measured in a biological sample of the patient, e.g. serum or plasma. Next, the antibody molecule is administered and then, after a period of time sufficient to allow the therapeutic antibody to exert its effect, the level of total IGF-1 is again determined. The amount of increase in the level of total IGF-1 compared to the level of total IGF-1 measured in the first step, indicates to which extent the patient responds to said anti-IGF antibody molecule. This method is preferably used for monitoring therapies in which the antibodies of the invention are administered.